The Optimal Designation And Application Of DNAzyme&the Synthesis And Application Of Novel Fluorescent Probe

Aptamers are single-chain nucleic acids （DNA or RNA） that bind any given molecular target with specificity. They are selected via the systematic evolution of ligand by exponential enrichment （SELEX） process. Compared to antibody, aptamers have many useful characteristics. For example, they are easily synthesized, readily modified, thermo-stable and can be further engineered to perform complex functions upon binding. There are some aptamers with G-rich sequences can fold into a four-stranded structure known as G-quadruplex. The G-quadruplex can bind hemin to form a catalytic complex with the peroxidase activity. These catalytic complexes were called DNAzymes. The catalytic ability of DNAzymes was affected by many factors. In the presence of hydrogen peroxide （H₂O₂）, DNAzymes can effectively catalyze the oxidation of2,2-azinobis （3-ethylben-zothiazoline）-6sulfonic acid （ABTS）,3,3,5,5-tetramethylbenzidine （TMB）,2’,7’-hydrochloride fluorescent diacetate （H2DCFDA）, etc. So the DNAzymes have been widely used in molecular recognition, biological analysis, biosensor, etc. This paper studied the factors that affect the catalytic ability of DNAzyme, and constructed electrochemical hydrogen peroxide biosensor using DNAzyme.The mercapto compound such as glutathione, cysteine, and homocysteine paly an important part in many amino acids and proteins. They have bearing on several pathological and physiological processes. The separation and detection of mercapto compound received widespread concern. They were mainly detected by the following method including HPLC, colorimetric, fluorescence, chemiluminescence, and electrochemical detections. In this paper a novel fluorescent probe FL-S-S-FL was synthesized and used as fluorescent probe to detect DTT, cysteine and glutathione. Details are as follows:1、Twelve nucleic acids were designed as aptamers to form DNAzymes with hemin. Four compounds, ABTS, H2DCFDA, tyramine and TMB were selected as substrates to be catalyzed by these DNAzymes. The results shown that the number of G-strand layers, the hairpin-like DNA duplex, the number of the nucleic acid bases in the lateral loops and spacers of hairpin-like DNA duplex all affect the hemin-binding affinity with aptamers. Besides, the DNAzymes’catalytic activity was not only determined by hemin-binding affinity but also affected by the spatial structure of substrate. For these DNAzymes, ABTS could be a good substrate for colorimetric determination and tyramine is a good substrate for fluorimetric determination.2、Dopamine can form polydopamine film via self-polymerization. The electrochemical biosensors for hydrogen peroxide detection were constructed using polydopamine-entrapped G-quad r up lex-hemin-DNAzyme. Eight DNA were designed to investigate the influence of different sequences of DNA. The results showed that the DNAzyme biosensors have good response to H2O2, when DNAzyme was fixed to the electrode surface, its secondary structure was relatively fixed than those in the solution. Hence, the structure has little effect on its catalytic ability. Compared to the sensor constructed by hemin, DNAzyme show better catalytic performance. The detection limit for H2O2was8.010-7M, the linear range was8.010-6-2.8810-4M.3、A novel fluorescent probe FL-S-S-FL was synthesized by the reaction of FITC and cystamine dihydrochloride. Due to resonance energy transfer, the fluorescence intensity of the probe solution was very weak. In the presence of a mercapto group, which can cut off the S-S bond, thereby significantly enhanced fluorescence intensity was observed, and the intensity was linear to the concentration of mercapto compound in a certain range. The detection limit for dtt was1.010-6M. The linear range was3.010-6-9.610-5M; the detection limit for cysteine and glutathione was5.010-6M, the linear range was1.910-5-1.8910-4M.